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Enzyme conjugates for use as detoxifying agents

Inactive Publication Date: 2009-10-01
BOLDER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule comprisi

Problems solved by technology

Exposure to organophosphorus (OP) compounds in the form of nerve agents is an ever-increasing military and civilian threat.
This results in the accumulation of acetylcholine at cholinergic synapses, particularly in the brain and diaphragm, producing a crisis that can culminate in death by respiratory failure.
Although these treatment regimens are effective in preventing lethality from OP poisoning, they do not prevent the post-exposure incapacitation, convulsions, performance problems and even permanent brain damage.
Unfortunately, most enzymes such as the recombinant AChEs and ChEs have relatively short residence times in circulation, on the order of minutes in mice (Saxena et al., 1998).

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

In vitro Spectrophotometric Assay for BuChE Proteins

[0116]The kinetic constants (Km and Kcat) of the BuChE protein variants can be determined for the substrates butyrylthiocholine and acetylthiocholine (Sigma) using a variation of the Ellman method as described previously (Platteborze and Broomfield, 2000; Ellman et al., 1961). Enzymatic hydrolysis of the substrate generates free thiocholine which reacts with Ellman's reagent (DTNB) to produce a yellow chromophore. The reactions are set up in microtiter plates and monitored at 412 nm. Commercially available equine butyrylcholinesterase can serve as a positive control for the assay along with human recombinant wild type butyrylcholinesterase.

example 2

Cloning, Expression, and Mutagenesis of BuChE

[0117]Recombinant expression of BuChE has been reported in E. coli (Masson, 1990), microinjected, Xenopus laevis oocytes (Soreq et al., 1989), insect cell lines in vitro and larvae in vivo (Platteborze and Broomfield, 2000), in silkworms (Wei et al., 2000) and in mammalian COS cells (Platteborze and Broomfield, 2000), CHO cells (Masson et al., 1993; Lockridge et al. 1997) and in transgenic goats (Nexia, Inc.).

[0118]A cDNA for Human BuChE was amplified by PCR from human liver, fetal human liver, and human pituitary cDNA libraries (QUICKclone™ cDNA libraries, Clontech). Primers BB1136 and BB1137 (see Table I for list of oligonucleotide primer sequences) were used in the PCR with the cDNA libraries as templates, producing fragments ˜1.6 kbp in length (termed fragment 1136×1137), which were gel-purified. The 1136×1137 fragments were further amplified in two ways. In one method, the fragments were further amplified using primers BB1076, BB1134...

example 3

Cysteine PEGylation Optimization and In vitro Evaluation of PEG-Cys-BuChE Candidates

[0124]BuChE cysteine muteins that retain in vitro activity are good candidates for screening studies using a cysteine-reactive 20 kDa PEG-maleimide. The cysteine muteins must be partially reduced with dithiothreitol (DTT), Tris (2-carboxyethyl) phosphine-HCl (TCEP) or other disulfide disrupting agent, in order to expose the free cysteine for PEGylation and allow the PEGylation reaction to proceed efficiently. Typically a 5-fold molar excess of DTT for 30 min is sufficient. Excess DTT can be removed by dialysis. The reduced protein is reacted with various concentrations of PEG-maleimide to determine the optimum ratio. PEGylation of the protein can be monitored by a molecular weight shift using SDS-PAGE. The lowest amount of PEG that gives significant quantities of mono-PEGylated product is considered optimum (80% conversion is considered good). The PEGylated protein is purified from the unPEGylated pr...

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Abstract

Disclosed are detoxifying enzyme conjugates, including conjugates of variants of such detoxifying enzymes. The detoxifying enzymes are preferably chlolinesterases, and more preferably, butyrylcholinesterase. Also disclosed are methods of making and using such conjugates.

Description

FIELD OF THE INVENTION[0001]The present invention generally relates to variants and conjugates of detoxifying enzymes and methods of making and using such variants and conjugates.BACKGROUND OF THE INVENTION[0002]Exposure to organophosphorus (OP) compounds in the form of nerve agents is an ever-increasing military and civilian threat. OPs were first developed in the 1930's for use as insecticides. Their potency was recognized during World War II, and they were developed as nerve agents for use in chemical warfare. The acute toxicity of OPs is usually attributed to their irreversible inhibition of acetylcholinesterase (AChE). This results in the accumulation of acetylcholine at cholinergic synapses, particularly in the brain and diaphragm, producing a crisis that can culminate in death by respiratory failure. Current treatment for OP poisoning consists of a prophylatic dose of a spontaneously reactivating AChE inhibitor to protect ACHE from irreversible inhibition of OPs and post-expo...

Claims

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Application Information

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IPC IPC(8): A01K67/00C12N9/16C07H21/00C12N5/00C12N5/06C12N1/20A61P39/00A01H5/00
CPCC12N9/18A61P39/00
Inventor ROSENDAHL, MARY S.
Owner BOLDER BIOTECH
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